The double stranded RNA-activated protein kinase, PKR, is an interferon (IFN) -regulated host defense enzyme essential for higher vertebrates to suppress the spread of different viruses. PKR is a key regulator of protein synthesis and gene transcription. PKR phosphorylates the protein synthesis initiation factor, eIF2alpha regulating its involvement in mRNA translation. In response to specific signals, it also mediates phosphorylation of important transcriptional regulators, including the inhibitor of NF-kappaB, IkappaB, and the transcription factor IRF-1. The long term objectives of this study are to understand how PKR functions at the molecular and cellular level to control viral infections and cellular homeostasis. To probe the mechanisms of viral or cellular dsRNA activation and inhibition of PKR, the three dimensional structure of PKR double stranded RNA binding domain (dsRBD) and its complex with the PKR activator, HIV-1 TAR RNA, will be determined using multidimensional heteronuclear NMR spectroscopy. Because NMR spectroscopy, is performed in solution, dynamic information about the structure of PKR and its interactions with TAR RNA will be obtained. To investigate the physiological role of PKR as an antiviral and pro- apoptotic protein, apoptosis will be studied in normal cells and in genetically altered cells and mice where PKR has been homozygously inactivated or the expression altered through transgenesis. Antiviral and apoptotic responses will also be measured in mice which have both major IFN-regulated pathways, PKR and RNaseL, deleted. Molecular partners which mediate PKR-dependent apoptosis will be identified and characterized. To establish the role of PKR in the regulation of cytokine biosynthesis and cellular homeostasis, mechanisms of Th1 and Th2 switching the Pkr percent mice will be investigated and the ability of these mice to mobilize defense systems against different viruses determined. Mechanisms will focus on the role of PKR interacting proteins. The regulation of PKR by dsRNA provides a unique means of activation of an antiviral enzyme to control viral infections. An understanding of the mechanisms of activation and of downstream targets of PKR may lead to the discovery and development of novel therapeutics for viral infections, cancer and immune dysfunction.